Electro-Optic Grating-Coupled Surface Plasmon Resonance (EOSPR)

ABSTRACT

An instrument for measuring and analyzing surface plasmon resonance (SPR) and/or surface plasmon coupled emission on an electro-optic grating-coupled sensor surface is described herein. The sensor chip achieves SPR through a grating-coupled approach, with variations in the local dielectric constant at regions of interest (ROI) at the sensor surface detected as a function of the intensity of light reflecting from these ROI. Unlike other grating-based approaches, the metal surface is sufficiently thin that resonant conditions are sensitive to dielectric constant changes both above and below the metal surface (like the Kretschmann configuration). Dielectric constant shifts that occur as mass accumulates on the surface can be returned to reference intensities by applying voltage across the underlying electro-optic polymer. Approaches to the development of the sensor surfaces are described, as are software and hardware features facilitating sample handling, data gathering, and data analysis by this solid-state approach.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/900,548 for “Electro-Optic Grating-Coupled SurfacePlasmon Resonance (EOSPR)”, filed Nov. 6, 2013, the disclosure of whichis incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This application is based on research and development done underNIH/NIGMS Grant No. 1R43GM104636-01.

TECHNOLOGICAL FIELD

The described embodiments relate to an instrument and method for thedetection, measurement, and characterization of a wide variety ofspecific molecules, simple or complex solutions, and biological cells.

BACKGROUND OF THE INVENTION

Requirements for the identification and measurement of biological orchemical entities in a sample typically include a means of isolating orseparating the analyte in question. Once spatially, temporally, orotherwise isolated from the surroundings, the analyte provides orpromotes a signal whose intensity can be interpreted as a measure of thepresence and/or concentration of analyte originally present in thesample. For example, one common approach to enzyme-linked immunosorbantassays (ELISAs) utilizes capture ligands to retain the analyte inquestion at the bottom of a microtiter plate while irrelevant moleculesare removed from the well in a series of washes. When afluorescently-labelled antibody targeting a second epitope isintroduced, the signal from adherent fluorophores can be interpreted asproportional to the concentration of analyte present in the originalsample. In this and other embodiments, absolute measurements areachieved via comparison with a parallel signal arising from a knownquantity of analyte.

Surface plasmon resonance (SPR) assays are based on the coupling ofP-polarized incident light to a surface plasmon wave, a physical modecreated by charge density oscillations at a metal-dielectric interface.The mismatch in dielectric constants between these two materialssupports a mode of evanescent electron excitation known as a surfaceplasmon. Coupling of the light to this mode, known as resonance,requires a momentum match between the plasmon and the incident light,and consequently is exquisitely sensitive to the properties of thelight. When conditions are optimal, the majority (>90%) of the lightacts to excite the plasmon, with little being reflected. Detectorsplaced in the optical path of reflected light are able to measure thisintensity as a function of incident angle, wavelength, or phase, andinstrumentation has been developed that generates intensity plots as afunction of each of these variables. The nadir of the intensity plot isknown as the SPR minimum, and it defines the conditions where optimalcoupling into the plasmon mode occurs.

The value of the independent variable defining the SPR minimum changeswith the dielectric constant at the surface, a phenomenon whichunderlies the utility of SPR techniques in biological or chemicalsensing. Accumulating mass (e.g. protein binding) at the sensor surfaceleads to a proportional change in the dielectric constant, and aconsequent change in the value associated with the SPR minimum. This“SPR shift” acts in a sense like a “molecular scale,” providingquantitative measures of changes in mass at the sensor surface. Thus SPRacts in a sense like a “molecular scale,” where change in surface massrecorded as an “SPR shift.” Detection of a specific biological moleculecan occur with the attachment of an appropriate capture ligand to thesensor surface, (antibodies, oligonucleotides, aptamers, etc.) servingto capture the analyte of interest present in a sample as it flows overthe surface. Additional flow of buffer provides a wash sequence toremove non-specific adherents. While this approach has much in commonwith ELISA, a secondary antibody is not required to measure the SPRsignal; the accumulation of mass is theoretically sufficient. Alas, SPRhas not proven sensitive enough to detect trace quantities of smallmolecules, particularly in complex media, and the low sensitivity ofclassic approaches to SPR as compared to labeled techniques like ELISAhas proven to be a limitation to the application of this technology toenvironmental and clinical samples.

SPR is an evanescent phenomenon and therefore the effect on thedielectric constant is limited to the region immediately surrounding themass. Indeed in at least one instrument architecture, densitiesexceeding 1,000 spots/cm² have been achieved with no significantcross-talk. This opportunity for spatial separation of multiple analytesutilizing an assortment of capture ligands permits an increase in thenumber of experiments performed on a single chip. When spotted in a 2Darray, parallel intensity measurements can be performed using a camera,with pixel clusters defined in silico around each antibody spot. Theseregions of interest (ROI) serve as independent assays of analytespresent in the sample, with shifts in the SPR minima of each ROIproviding binding kinetics and end-point concentrations.

The Kretschmann Configuration—The most common method for momentummatching between incident light and the surface plasmon mode is known asthe Kretschmann configuration and is dependent on the sensor surfacebeing in optical contact with a high-refractive index prism. Limitationsof the Kretschmann configuration include instrument size, instrumentprice, ease of use, and the relatively small number of simultaneousanalyses that can be performed. The physics of this configuration as anSPR detection scheme is well-established (see Homola, J., 2006, “SurfacePlasmon Resonance Based Sensors”, Springer Series on Chemical Sensorsand Biosensors: Methods and Applications: 1-252), but for the purpose ofthis disclosure one key element of the design should be noted.Kretschmann instrumentation architecture most frequently contains asingle metal layer with two pertinent surface interfaces, to be knowngoing forward as the metal-sample interface and the metal-prisminterface. In this configuration, coupling is dependent not only on thedielectric constants of the metal and the sample, but also on that ofthe prism as can be seen in equation 1:

$\begin{matrix}{{\tan \left( {\kappa_{sp}{d/2}} \right)} = \frac{{\gamma_{1}{ɛ_{2}/\kappa}\; ɛ_{1}} + {\gamma_{3}{ɛ_{2}/\kappa}\; ɛ_{3}}}{1 - {\left( {\gamma_{1}{ɛ_{2}/\kappa}\; ɛ_{1}} \right)\left( {\gamma_{3}{ɛ_{2}/{\kappa ɛ}_{3}}} \right)}}} & (1)\end{matrix}$

where κ_(sp) is momentum of the plasmon wave that exists on adielectric-metal-dielectric and κ²=ω²∈₂∈₀μ₀−β² and γ_(1,3)²=β²−ω₂∈_(1,3)∈₀μ₀. d is the thickness of the metal layer, ω is theangular frequency, ∈₀ is permittivity of free space and ∈_(n) is thedielectric constant of medium n, μ₀ is the free-space permeability, andβ is the propagation constant of the plasmon mode. In the Kretschmannconfiguration, ∈₁ is the dielectric constant of the sample, ∈₂ is thatof the metal, and ∈₃ is that of the prism. The metal layer is madesufficiently thin (˜50 nm) that changes in the resonant conditionsestablished by sample accumulation at the metal-sample interface areaffected by the dielectric constant of the prism at the metal-prisminterface. This reduced thickness permits the plasmon to “penetrate”into the prism, and allow for coupling conditions to be interrogatedfrom the opposite side of the metal film as the sample.

Grating-coupled SPR (GCSPR)—This approach achieves momentum matchingusing diffracted light, often produced by means of a sensor chip with anembossed diffraction grating. This coupling scheme simplifies samplepreparation, reduces instrument cost, and allows for epi-illuminationoptics, vastly increasing the number of assays performed simultaneously.Although detection of the SPR minimum in a GCSPR system can be readilyachieved by varying angle, wavelength, or phase, this discussion willfocus on an angle-scanning approach to GCSPR. Similar principles applyfor these other detection modalities, but an angle-scanning approachsimplifies equations and facilitates direct comparison with commerciallyavailable technologies. In these platforms, much like their Kretschmanncounterparts, changes in dielectric constant due to bound mass (Δ∈₁)affect κ_(sp), while the values of ∈₂ (the metal) and ∈₃ (the underlyingsubstrate) do not vary. Similarly, in GCSPR, the change in κ_(sp) isdetected by varying the incident angle (θ) until the light's momentum(k-vector) matches κ_(sp), and resonance is achieved. The equation thatgoverns coupling into the GCSPR system is reproduced in equation 2:

κ_(source) sin θ+mκ _(grating)=κ_(sp)(∈₁,∈₂,∈₃)  (2)

The above shows that when κ_(source) and κ_(grating) are fixed, asoccurs readily in classic GCSPR instruments, the mass-dependent changesin ∈₁ shape κ_(sp) and are observed as changes in the coupling angle(θ). Local refractive index changes at the metal-dielectric interfaceare detected with incident light that passes through this dielectric, soin this configuration, the thickness of the metal is irrelevant, as longas it exceeds the minimum necessary to support SPR. Due to therobustness and the relative ease of manufacture, metal layers in mostGCSPR platforms have been sufficiently thick (˜1 μm) as to obscure anyeffect of ∈₃ on the plasmon coupling conditions. While GCSPR systemsoffer considerable improvement over the limitations of Kretschmannsystems, they remain less sensitive than fluorescent techniques, and thedependence on moving parts for angle scanning makes these instrumentsrelatively fragile and therefore ill-suited for field use.

Surface Plasmon-Coupled Emission (SPCE)—It has been observed that energyfrom surface plasmons can be out-coupled and absorbed by fluorophoremolecules in close proximity to the metal surface (see Lackowicz, J. R.,2006, “Plasmonics in Biology and Plasmon-Controlled Fluorescence”, DOI10.1007/s 11468-005-9002-3). The local field of the propagating wave atthe metal/dielectric boundary enhances absorption of plasmons ascompared to free-space absorption. The subsequent fluorescent emissionis out-coupled into propagating lobes in accordance with the momentummatching conditions previously described. Fluorescence generated in thismanner is emitted as directional lobes rather than omnidirectionally asin a solution (i.e., as in a typical fluorimeter). An optical detectionsystem can be designed to capture this SPCE with much greater efficiencythan can be done with omnidirectional fluorescence. This enhancedcapture efficiency results in considerably greater detection sensitivityand is sufficient to quantitatively measure cytokine secretion fromsingle cells (see Reilly, M. T., et al., 2005, “SPR surface enhancedfluorescence with a gold-coated corrugated sensor chip” Progress inBiomedical Optics and Imaging—Proceedings of SPIE Volume 6099, Articlenumber 60990E DOI: 10.1117/12.646165).

Electro-optic polymers are polymers with non-linear optical properties,such that they change their dielectric constant and refractive index asa function of an applied electric field. Polymers displaying thePockel's or Kerr effect in response to applied voltage have beendescribed and are in use in high speed optical switches in thetelecommunications industry. The considerable thermal, chemical, andtemporal stability are a sine qua non for commercial optical switches,and this patent aims to take advantage of recent advances enablingnanoscale patterning of said polymers.

BRIEF SUMMARY OF THE INVENTION

An analytical sensor platform that facilitates the capture and detectionof specific cells or molecules on the surface of an electro-opticgrating-coupled surface plasmon resonance “chip” is described herein.The complete platform is comprised of a sensor chip with an integratedor stand-alone yet complementary detection apparatus supporting surfaceplasmon resonance and/or surface plasmon coupled emission analyses. Asdetailed below, one sensor chip architecture consists of an inertsubstrate for structural support, upon which a conductive layer isdeposited, upon which a grating-embossed or otherwise patternedelectro-optic polymer is deposited, and upon which a thin gold layer isplaced. In this embodiment, each of the two conductive layers is inelectrical contact with contact pads, which interface with asoftware-controlled voltage generator.

Instrumentation will consist of, at minimum, a source of polarized,collimated light suitable for achieving SPR and/or SPCE on the proposedchip. The chip will be manufactured to include or converted by theend-user into a fluid-tight enclosure known as a “flow chamber,”permitting the introduction and retention of samples, buffers, or otherreagents. One design forms a flow chamber by means of a gasketsurrounding the active area of the chip and a transparent windowsandwiching the gasket and establishing a small volume for liquids topass over the sensor chip. Fluid inlets and outlets manifesting as smallholes in the window will interface with pumps, valves, tubing, and otherfluidic systems necessary to provide circulation or recirculation ofsample, wash, or other fluid over the active area of the sensor chip.Optics will act to isolate signal from noise and project emitted lightonto a director surface (e.g. camera, photodiode array), and whenmeasuring SPCE, direct incident light away from the detector, maximizingthe contribution of fluorescent emission to the signal. Computersoftware will permit the manual or automated control of optics,fluidics, and voltage; and will also assist with data collection,manipulation, analyses, and presentation.

The EOSPR chips will present development challenges, and we seek todescribe herein one method of chip manufacture. Briefly and asenvisioned, this manufacturing effort will consist of deposition of aconductive material onto a structural support platform, positioned belowwhat will become the active area of the chip, with a small extensionfacilitating electrical interfacing with this layer. Overlying thislayer, at the active area of the chip, will be a layer of EO materialwhose dielectric and optical properties vary with the strength of anelectric field applied to the EO material. One example of an EO materialis a poled EO polymer patterned to form a diffraction grating. Twopotential approaches to this step are presented below to illustrate thegeneral concept. The first will deposit the polymer as a uniform layer,poling the polymer as it relaxes below its glass temperature. Thegrating structure will subsequently be generated directly in the polymerusing lithographic patterning techniques. The latter approach willemboss hot polymer with a metal shim containing a complementarydiffraction grating pattern. One example of an EO polymer materialcompatible with the disclosed EOSPR chips is SEO100 electro-opticpolymer from Soluxra, LLC of Seattle, Wash.

In one disclose example, poling will occur as the polymer cools with anelectric field applied between the “stamp” and the lower conductivelayer of the chip. In either case, an SPR-active material is depositedon top of the grating. The most common SPR-active materials are metalswith free electrons available for coupling into surface plasmonspropagating along the surface of the metal. The SPR active metal will bedeposited atop the grating as a thin layer, and will also extend to ablank region of the chip to facilitate the application of a voltagebetween the two conductive layers sandwiching the EO polymer toestablish an electric field in the EO polymer.

As with many other SPR platforms, the variable representing theaccumulation of mass at the sensor surface is the SPR minimum ofreflected intensity at individual ROIs. Unlike most other SPR platforms,electro-optic surface plasmon resonance observes the intensitymodulations defining an SPR minimum as a function of a voltage appliedacross an electro-optic polymer. The notion of an electro-optic approachto SPR is not without precedent; cf. U.S. Pat. Nos. 6,667,807,8,009,356, but the novelty and advantages of the claimed detectionschema absolutely require the combination of this technology with agrating-coupled approach to SPR.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic drawing showing one possible optical path for theinstrument; and

FIG. 2 shows several views of one possible EOSPR chip.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “electro-optic polymer” or “EO polymer” refersto polymers or other materials whose dielectric constant varies as afunction of applied voltage. SEO100 from Soluxra, LLC of Seattle, Wash.is an example of an electro-optic polymer potentially compatible withthe disclosed embodiments. The acronym GCSPR stands for Grating-CoupledSurface Plasmon Resonance, and EOSPR stands for Electro-Opticgrating-coupled Surface Plasmon Resonance. SPCE stands for SurfacePlasmon Coupled Emission. The EOSPR sensor surface is alternativelycalled the “chip,” the “sensor chip,” or the “EOSPR chip,” and willsupport an electro-optic grating-coupled approach to both SPR and SPCE.

An EOSPR sensor chip and complementary detection schema providingsurface plasmon resonance analysis with or without concomitant SPCEmeasurements are described below.

In a dielectric-metal-dielectric arrangement, Equation 1 holds true, andκ_(sp) therefore depends on the dielectric constant of all three layers(∈₁, ∈₂, and ∈₃). In the Kretschmann configuration, local changes in thedielectric constant due to bound mass (Δ∈₁) affect κ_(sp) while thevalues of ∈₂ and ∈₃ do not vary. The change in κ_(sp) in theseinstruments is detected by adjusting the momentum of the source light,often by varying the incident angle (θ) until resonance is achieved. Inthe grating-coupled approach, coupling into the GCSPR system is governedby Equation 2. When κ_(source) and κ_(grating) are fixed, as occursreadily in classic GCSPR instruments, the mass-dependent changes in ∈₁shape κ_(sp) and are observed as changes in the coupling angle (θ).Equations that define κ_(sp) and the coupling angle θ for Kretschmanninstruments are similar. However, the EOSPR platform departs from thesedetection schema by restricting θ to a given angle (e.g. the SPR anglefor a water-gold-plastic construct), or a small range of angles andinstead interrogating accumulation or depletion of bound mass bychanging ∈₃. To clarify, one approach to detection would measure theeffect of accumulating mass (manifesting AO not by changing theproperties of the incident light (e.g. angle), but instead by changingthe properties of the underlying grating itself (Δ∈₃). As alluded toabove, classic GCSPR instruments are not thought of asdielectric-metal-dielectric systems because the metal layer is typicallythick enough to obscure any impact of ∈₃. For ∈₃ to be relevant, as isproposed in this EOSPR approach, the thickness of the metal layer mustbe reduced to thicknesses such as those found in Kretschmann e.g., lessthan approximately 50 nm. In this case, the controlled change in ∈₃ willbe accomplished by replacing the underlying dielectric with a polymerdisplaying a significant electro-optic (EO) effect.

Integrating an EO polymer into a GCSPR-style chip permits a detectionscheme where departures from resonant conditions (Δ∈₁) will be measuredsolely by varying the dielectric constant of the EO polymer. Thedielectric constant change at the surface due to binding mass (typicallyno larger than ˜10⁻⁵ RIU) is readily matched by changes in thedielectric constant of the polymer (which can modulate by as much as10⁻³ RIU). The precise match between incident light and the plasmon modewould therefore not be a function of angle, but a function of appliedvoltage.

One embodiment of the EOSPR chip (20) is sketched as FIG. 2, thecomponents of which are built upon an inert substrate (21). Above thissubstrate is a conductive layer (25) whose properties support theapplication of voltage and adhesion of the polymer layer (26). Thisconductive layer need not be metal nor SPR active, but along with theSPR-active metal surface (24) acts to promote the application of auniform electric field across the EO material. The EO material (26) is alayer that has been patterned to act as a diffraction grating, but whoseoptical properties continue to display a significant electro-opticeffect. An SPR-active metal surface (24) overlies this particularembodiment, and contact pads (22 and 23) are continuous with the twoconductive layers (24 & 25).

The instrumentation portion is presented here as a standalone deviceinterfacing with the EOSPR chip, providing control of input voltage,monitoring reflected light intensity at the sensor surface, controllingfluidics, and interpreting data. Devices offering any and all of thesefunctionalies in a package designed to support this grating-coupledelectro-optic approach to SPR or SPCE sensing shall comprise an “EOSPRinstrument” for the purposes of this discussion. As presented, theminimum components for such a system include a light source (30), apolarizer and collimated lens (31), the chip (20, perhaps positioned ona removable holding apparatus, 35), suitable filters and lenses (32), adetector (33), the voltage generator (34), and a computer. Fluidics,their interfaces, and controls may be a component of the chip, acomponent of the instrument, or may stand alone, but are not representedin the figures for clarity.

The archetypal detection scheme proposed herein involves optimizing theinstrument so that the incident angle and the detector (camera,photo-diode array, etc.) are fixed at approximately the SPR angle forthe bare gold surface. Room for adjustment of this angle can beengineered into the design, permitting user calibration and/or increasedinstrument tolerance for varied environmental conditions. Instead ofmonitoring reflected intensity as a function of incident angle, theinstrument would measure reflected intensity as a function of voltageapplied to the bottom electrode. In the most straightforward scanningprotocol, the incident angle and the location of the light detector willnot move. Since electro-optic polymers change dielectric constantpredictably in the presence of an electric field, applying a voltage tothis basal electrode while maintaining the surface metal at ground wouldgenerate a change in the dielectric constant of the sandwiched polymer(Δ∈₃). With the surface grounded, interference with biologicalinteractions is not expected. The assay would monitor the binding ofmass to the surface by measuring the voltage required to return all ROIto resonance or a reference value. The changes in local dielectricconstant imparted by the bound mass on the surface would be in essencenullified by changes in the dielectric constant in the polymer layer.Several related works appear in the literature (including prior art fromCiencia), but none appear as well suited for commercial adoption as theEOSPR platform, primarily due to the epi-illumination architecture.

The addition of an electro-optic layer in between conductive layers addsslight complexity to the chip, yet greatly reduces the requirements forinstrumentation. The EOSPR chip eliminates the need for moving parts andsignificantly shortens the optical pathway. The proposed device would beable to simultaneously measure the shifts for the same number of ROI(˜1,000/cm²) as allowed by extant GCSPR instrumentation. The number ofspots is constrained by the active area of the chip, since the densityof spots is limited to prevent an overlap of plasmon waves. There is nofundamental reason why smaller or larger chips with tens or millions ofspots could not be developed for future instruments. In anyimplementation, the reduced weight, cost, and fragility would make EOSPRinstrumentation more portable and affordable, while increasingsensitivity over comparable GCSPR platforms. By selecting smallcomponents and a smaller chip (active area of ˜4 mm×4 mm, enough for˜100 spots) we have calculated that an optimized and sensitive EOSPRinstrument could be about the size and weight of an average hardcovernovel. Bringing a sensitive and high-content assay into the realm ofhand-held and battery-operated devices invites enticing marketopportunities.

Although it is hard to quantify the sensitivity increase expected fromthis design ab initio, several factors inherent to this system implythat the inclusion of EO polymers will boost instrument performancebeyond today's standards. Implicit in the EOSPR design are increases inthe quality of the signal and decreases in the noise compared to otherSPR platforms. Applied voltage can be measured more accurately than themechanical changes of incident angle present in current systems, thuslocating the SPR with high precision. In addition, thousands ofmeasurements defining the SPR minimum could be conducted in the time ittakes to perform a single scan on current instrumentation. Thisincreased scanning velocity would also permit direct measure of fasterreaction kinetics. Finally, voltages could be applied in a pseudo-randomfashion with collected data interpreted in silico, thus reducingsystematic error in measurements.

Besides boosting the quality of the signal, direct reduction ofinstrument noise is possible with such a rapidly scanning instrument.Even with an inexpensive 30 Hz camera, we can employ a signal-choppingscheme to subtract noise as background. Essentially, the voltage appliedto the EO material would switch from a near-resonant voltage to anoff-resonant voltage at a rate of 30 Hz synchronized to the camera. Thenear-resonant image provides the signal, while the off-resonant imageprovides the background. The difference between these two images wouldbe immune to many sources of noise, such as stray light, changes intemperature, changes in light intensity, etc. For a 30 Hz frame rate, 15background subtracted images can be obtained per second, still allowingfor extensive averaging at a reasonable data acquisition rate. Theenhanced quantities of high-quality data collected by the electronicdetection scheme and the expected reduction of noise encountered bychopping the signal combine to strongly suggest an overall increase ininstrument sensitivity. High-sensitivity measurements and a compact,stable, and no-moving-parts design strongly suggest this platform wouldbe ideal for field use.

What is claimed is:
 1. A sensor comprising: a substrate; a conductivelayer on said substrate; a layer of electro-optic material on saidconductive layer, said layer of electro-optic material including adiffraction grating on a surface opposite said conductive layer andhaving a dielectric constant and refractive index which vary as afunction of the strength of an electric field applied to saidelectro-optic material; and an SPR-active layer on said diffractiongrating, wherein voltage applied to said conductive layer and saidSPR-active layer establish an electric field in said electro-opticmaterial and changes in the applied voltage alter the strength of saidelectric field, thereby changing the dielectric constant and refractiveindex of said layer of electro-optic material.
 2. The sensor of claim 1,wherein said electro-optic material is an electro-optic polymer.
 3. Thesensor of claim 1, wherein said SPR-active layer is a metal layer lessthan approximately 50 nm in thickness.
 4. The sensor of claim 3, whereinsaid metal layer is gold.
 5. The sensor of claim 1, wherein saiddiffraction grating, electro-optic material, and SPR-active layer areselected to result in a known resonant angle at which collimated,polarized light of a pre-determined frequency couples to said SPR-activesurface and said resonant angle changes as a function of the strength ofsaid electric field.
 6. The sensor of claim 5, wherein said resonantangle changes in response to a mass of material bound to said SPR-activesurface and the applied voltage can be varied to change the strength ofsaid electric field to correct for changes in said resonant angle due tomaterial bound to said SPR-active surface and the change in voltage canbe used to measure said mass of material bound to said SPR-activesurface.
 7. An instrument for detecting changes in the opticalproperties of a sensor surface defining an image plane, said instrumentcomprising: an electro-optic material having a surface including adiffraction grating coincident with said image plane, said electro-opticmaterial having a dielectric constant and refractive index that changeas a function of the strength of an electric-field to which theelectro-optic material is exposed; an SPR-active layer on saiddiffraction grating; a voltage generator arranged to deliver a variablevoltage to said electro-optic polymer, thereby exposing saidelectro-optic material to a variable electric field which changes thedielectric constant and refractive index of said electro-optic material;a source of collimated polarized light of a predetermined wavelengtharranged to project said collimated polarized light onto said imageplane at a defined angle of incidence; an imaging detector arranged toform an image of said sensor surface from said polarized light reflectedfrom said sensor surface; wherein at least a portion of said polarizedlight is coupled into surface plasmons at said sensor surface and theintensity of the reflected light received by said imaging detectorvaries as a function of the intensity of said electric field.
 8. Theinstrument of claim 7, wherein said electro-optic material, diffractiongrating, SPR-active layer, and predetermined wavelength result in aresonant angle at which a majority of the polarized light is coupledinto surface plasmons at said SPR-active layer and said resonant anglechanges as a function of the strength of said electric field.
 9. Theinstrument of claim 8, wherein said resonant angle changes in responseto a mass of material bound to said SPR-active layer, the appliedvoltage can be varied to change the strength of said electric field tocorrect for changes in said resonant angle due to material bound to saidSPR-active layer and the change in voltage can be used to measure saidmass of material bound to said SPR-active surface.
 10. A method ofmeasuring changes in the optical properties at a sensor surface definingan image plane comprising the steps of: providing an electro-opticmaterial with a conductive layer on one side and a diffraction gratingopposite the conductive layer and an SPR-active layer on saiddiffraction grating forming said sensor surface, said electro-opticmaterial having a dielectric constant and refractive index that vary asa function of an electric field established between said conductivelayer and said SPR-active layer; connecting a variable voltage generatorto said conductive layer and said SPR-active layer; arranging a sourceof collimated polarized light of a predetermined wavelength to projectsaid collimated polarized light onto said sensor surface at a definedangle of incidence; positioning an image detector to form an image ofsaid sensor surface from said polarized light reflected from said sensorsurface; monitoring the reflected intensity as a function of a variablevoltage applied across said conductive layer and said SPR-active layer.11. The method of claim 10, comprising: establishing a reference imageof said sensor surface at a first voltage applied across said EOmaterial; causing material to bind to said sensor surface, therebyaltering the optical properties of the sensor surface; and varying thevoltage applied across said EO material to re-establish said referenceimage at a second voltage; and employing a change in voltage betweensaid first voltage and said second voltage to determine the mass ofmaterial bound to said sensor surface.
 12. The method of claim 10,wherein said step of connecting a variable voltage generator comprisesconnecting said SPR-active layer to ground.